The long-term goal is to define the biochemical and genetic mechanisms that regulate phosphatidylcholine (PtdCho) biosynthesis. The control of PtdCho production is central to membrane phospholipid homeostasis, and CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of the major route for PtdCho synthesis in mammals. PtdCho is the principal structural component of the membrane bilayer accounting for between 50 and 80% of the cellular membrane and is a precursor to the other two major membrane phospholipids. Historically, research has focused on the regulation of PtdCho synthesis as it relates to membrane biogenesis, a fundamental process common to all cells. However, there are many tissue-specific roles for PtdCho in animals that are essential components of mammalian physiology, but do not readily lend themselves to analysis in cell culture. The research plan for the coming grant period expands to encompass the construction of knockout mouse models to investigate the non-redundant roles of specific CCT isoforms in PtdCho synthesis in specialized tissues while continuing to unravel the molecular details of CCT regulation by lipids that is critical to the universal control of membrane homeostasis in all cells. The first aim will elucidate the unique functions of the CCT isoforms in the regulation of mammalian lipid metabolism. Our most significant recent contribution was the characterization of the number and distribution of CCT genes and enzymes in mammals. We have generated a CCTbeta2(-/-) mouse strain that exhibits unexpected phenotypes, and have a breeding colony of homozygous CCTalpha[flox] mice. We are assembling a collection of transgenic Cre recombinase mouse strains that will allow us to define the tissue-specific roles of CCT isoforms as well as determine if either CCTalpha or CCTbeta can be eliminated in the whole animal. We are in an excellent position to define the functions of specific CCT genes/proteins in mammalian physiology. The second aim focuses on the mechanisms by which lipids regulate CCT isoforms. Understanding how CCT senses lipid composition is central to appreciating the role of this enzyme in phospholipid homeostasis. We will critically test the hypothesis that curvature elastic stress is sensed by the CCT helical domain and evaluate the role of anionic lipid regulation via the C-terminus. The functions of the CCT regulatory domains characterized in vitro will also be evaluated for cellular localization and membrane binding in living cells.